Nuclear DNA Modal Patterns as Diagnostic Criteria in Human Lung Cancer

Transcription

1 Jpn. J. Clin. Oncol. 1981, 11 (3), Nuclear DNA Modal Patterns as Diagnostic Criteria in Human Lung Cancer HIROKI S. OZAK.I, M.SC, TAKESHI YONEYAMA, M.D.^ KENJI EGUCHI, M.D. : \ TETSURO KODAMA, M.D. 4 AND KAZUMASA HOSHINO, M.D. 1 Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 1, Department of Surgery-, Department of Internal Medicine-*, National Cancer Center Hospital, and Department of Pathology*, National Cancer Center Research Institute, Tokyo Abstract In a prospective study of human lung cancer, the nuclear DNA content of tumor populations on 76 touch smears prepared from primary and metastatic lesions of 70 patients was determined cytofiuorometrically. A modal DNA value was calculated from the distribution of the logarithmically transformed DNA content for each tumor. By the analysis of levels of modal DNA values, the nuclear DNA distribution histograms in lung tumors could be divided into the following three major types: diploid-tetraploid type (Type I), triploid type (Type II), and mixed type (Type III). Modal DNA values in Type I histograms were diploid (2c), tetraploid (4c), and octaploid (8c), whereas the modal DNA values in Type II histograms were near-triploid (3c), nearhexaploid (6c), and decaploid (10c). Type III histograms were characterized by the simultaneous existence of multiple modal DNA values at diploidy or tetraploidy and near-triploidy or near-hexaploidy. Each of the three major types was further divided into six, four, and four kinds of DNA histogram subtypes, respectively, according to modal DNA values. The most frequent DNA distribution pattern was the subtype A of the triploid type (Type IIA). Four of 14 DNA histogram subtypes were all of polyploid distribution patterns in which the relative DNA values in the individual modes differ from each other by a factor of two. This suggests that endoamitosis accounts for such polyploidy. In one of five patients whose primary and metastatic lesions were examined simultaneously, the DNA distribution pattern changed from Type IIA in the primary tumor to Type IIB in its metastasis. The diagnostic and prognostic implications of the 14 DNA histogram subtypes analyzed are discussed. Introduction found to deviate from that of normal control tissue (Bohm and Sandritter, 1975). In the last decade, the nuclear DNA Some efforts have been made to utilize the distribution pattern in malignant tumors was results analyzed from such deviations for diagnostic (Bohm and Sandritter, 1975) Received June 19, and even for prognostic (Atkin, 1972; Reprint requests: Hiroki S. Ozaki, Depart- Atkin and Kay, 1979; Caspersson, 1979; ment of Anatomy, Faculty of Medicine, Kreicbergs et al., 1980) evaluation of malig- Kyoto University, Sakyo-ku Kyoto 606, Japan nant neopi asia ReC ently, a distinct correla- This work was supported by Grants-in-Aid,,. from the Ministry of Health and Welfare tlon between the occurrence of certain types (54-12) and Yamada Science Foundation. of DNA distribution histograms and the

2 432 OZAKI et al. Jpn. J. Clin. Oncol. December 1981 grade of malignancy of breast carcinomas was demonstrated (Auer et al., 1980). It seems possible to obtain prognostic information by determining the DNA histogram type, in the individual case, as a supplement to the findings furnished by clinical staging, histopathological classification, and malignancy grading (Auer et al., 1980). A number of cytophotometric studies of lung tumors (Adams and Dahlgren, 1968; Bohm and Sandritter, 1975; Greisen, 1971; Nasiell et al., 1978) have been reported since quantitative measurements of nuclear DNA content were claimed to be useful as a diagnostic tool (Atkin, 1964; Wied et al., 1966). However, the analysis and classification of DNA distribution histograms by means of modal DNA values have never been undertaken in lung cancer. In the present study, an attempt was made to divide the nuclear DNA distribution histograms in lung cancer into some possible types by the analysis of modal DNA values, which were calculated from the distribution of logarithmically transformed DNA content. Source of Tumor Materials and Methods The 76 lung tumor samples, consisting of 66 primary and 10 metastatic lesions, used in the present study were all surgical specimens obtained from 70 patients in the National Cancer Center Hospital, Tokyo. There were five patients with both primary and metastatic lung tumors at the time of surgical operation. By stamping a freshly cut surface of unfixed tumor tissue on carefully cleaned and defatted nonfluorescent glass slides, touch smears were prepared and immediately wet-fixed. Histopathological diagnosis of each lung tumor was made on paraffin sections of the same specimen. The diagnoses of the 76 lung tumors were 48 adenocarcinomas, 14 squamous cell carcinomas, four adenosquamous carcinomas, five large cell carcinomas, two small cell carcinomas, two carcinoids, and one mucoepidermoid carcinoma. Fixation Smears were fixed in a fluid consisting of 85:10:5 volume percent methanol: formalin: glacial acetic acid (Bohm et al., 1968) for 1 hr. After fixation, smear preparations were briefly dehydrated in 96% ethanol and stored in the dark up to 2 wk until use. Cytochemistry Smear preparations were hydrolyzed in 5 N HCI for 8 min at 25 C and stained with acriflavin SchifFs reagent (0.001%, 10 min, 7 C) for the Feulgen reaction. In order to block the nonspecific dye-binding of acriflavin, smears were treated with azocarmin G (0.002%, 4 min, 25 C) prior to hydrolysis (Takamatsu et al., 1980). Quantitative Measurement of Nuclear DNA Content The Feulgen DNA content in each nucleus was quantitatively determined at 520 nm by a cytofluorometer (Olympus, Model BH-MSP-K) interfaced with a YHP 9845B desk-top computer (Yokogawa Hewlett Packard). Generally, more than 200 cancer nuclei and at least 30 control nuclei were randomly selected for measurement in each case. Tumor cells were readily distinguishable from normal control cells by nuclear morphology and the appearance of chromatin condensation. Diploid leukocytes and morphologically normal lung epithelial cells present in the smear preparation were used as controls. In order to compare the cytofluorometric results obtained from different specimens, the mean DNA content of normal diploid cells in each smear was designated as 2c, and then the DNA content of tumor cells was expressed in an arbitrary 2c unit. Modal DNA values (DNA ploidy of a tumor stem line) were analyzed from the distribution of logarithmically transformed

3 Vol. 11, No. 3 DNA HISTOGRAM TYPES 433 % 15 O 10 z o 2C 4C 6C D N n n nt 8C IOC I2C I4C 2C 4C A Fig. 1: A: Distribution histogram of nuclear DNA content constructed in an arbitrarily chosen interval. The stem mode (M) is discernible in the neartriploid region, whereas the mode of replicated DNA is not so distinct. B: After a logarithmical transformation of the data in Fig. I A. Note symmetry of (M) and (M X 2) distributions. The (M X 2) mode is now well depicted in the near-hexaploid (6c) region. DNA content for each tumor (Fig. 1). Results By analysis of the DNA modal patterns, the nuclear DNA distribution histograms from 76 lung tumors could be divided into three major types: diploid-tetraploid type (Type I), triploid type (Type II), and mixed type (Type III). Each of the three major types was further divided into six, four, and four different DNA histogram subtypes, respectively, according to the modal DNA values. Diploid-tetraploid Type (Type I) D N 8C I6C Type I tumors are characterized by heteroploid cell populations with only diploid (2c), tetraploid (4c), and octaploid (8c) DNA content (Fig. 2). Modal DNA values of the six subtypes in Type I are shown in Table 1. Type IA is a unimodal diploid distribution pattern with a few cells at 4c, which is indistinguishable from that of normal control tissue. This DNA distribution pattern prevailed exclusively in low-malignant lung tumors (2 carcinoids and 1 mucoepidermoid carcinoma). These tumors usually show no or low proliferative activity. Type IB populations show a bimodal DNA histogram with a basic DNA value in the diploid region and a concomitant duplication peak in the tetraploid region. Some cells with DNA values corresponding to the DNA synthesis phase of normal control cells and a few cells with DNA content at from hypertetraploidy to octaploidy are present. Type IC is a polyploid distribution pattern with two concurrent peaks around the diploid and tetraploid regions. A duplication peak of the 4c tumor cell population is seen in the octaploid region. Type ID is a trimodal distribution pattern with a main peak in the tetraploid or near-tetraploid region. Two minor peaks are recognizable in the diploid and octaploid regions, the peak in the latter region being a duplication of the 4c stem line. There are a few nuclei at 16c. Both Type IE and IF are tetramodal DNA

4 434 OZAKI el al. Jpn. J. Clin. Oncol. December 1981 % 30 TYPE I LL NO. LU O % n % 20 i n n B Jb. L Jl \_h GL 10- kdl 2C 4C 8C D N A 2C 4C 8C I6C 32C D N A Fig. 2: DNA distribution patterns of the six subtypes in Type I (diploidtetraploid type).

5 Vol. 11, No. 3 DNA HISTOGRAM TYPES 435 histograms with four peaks in the 2c, 4c, 8c, and 16c regions. Type IE has two modal DNA values in the tetraploid and octaploid regions, whereas Type IF shows a distinct modal value in the octaploid region. In both Type IE and IF histograms, a minor peak in the 16c region is a concomitant duplication of the 8c stem line. Triploid Type (Type 11) DNA Type IA IB IC ID IE IF Table 1 Modal DNA Values in Type 1 Histograms Modal DNA Values (2c units) & 3.8 & 2.2 & & Modal DNA values in triploid type histograms are near-3c, near-6c, and 10c (Table 2). The four different DNA distribution patterns in Type II are shown in Fig. 3. Type IIA shows a bimodal DNA histogram with a distinct modal value in the near-triploid region and a concomitant duplication peak in the near-6c region. Usually there are only a few diploid and tetraploid cells in Type IIA histograms, but in some cases a tumor subpopulation and its duplication peak are found around the diploid and tetraploid regions, respectively. Type 1IB is a polyploid distribution pattern with two well-defined peaks around the near-3c and near-6c regions. Two concurrent peaks in the Type IIB histogram differ from each other in their DNA values by a factor of two (Table 2). A duplication peak of the stem line with near-6c value is seen Histopathological Diagnosis Mucoepidermoid ca. Carcinoid Carcinoid Adenosquamous ca. Small cell ca. Adenosquamous ca. Small cell ca. Large cell ca. Large cell ca. I C\C Q \ 1 f\ fl Case No

6 436 OZAKI et al. Jpn. J. Clin. Oncol. December 1981 in the near-12c region. Type IIC is characterized by a distinct modal DNA value in the 5c region. Two minor peaks are found in the hyperdiploid and near-10c regions, the peak in the latter region being a duplication of the stem line with near-5c value. The Type I1D histogram exhibits pronounced irregular aneuploidy, with DNA content ranging from near-2c to values beyond 16c or even 20c. However, two rather broad peaks are seen in the 5c and 10c regions. Mixed Type (Type III) Mixed type histograms are characterized by the simultaneous existence of two modal DNA Type IIA IIB Table 2 Modal DNA Values in Type II Histograms Modal DNA Values (2c units) & & 5.6 & 5.8 & 5.8 & 5.8 & & & & 6.4 Histopathological Diagnosis Adenosquamous ca. A denocarcinoma Large cell ca. Adenosquamous ca. LULallUIl Case No IIC A denocarcinoma IID 5.0 &

7 Vol. 11, No. 3 DNA HISTOGRAM TYPES 437 DNA values at diploidy or tetraploidy and near-triploidy or near-hexaploidy (6c) (Table 3). Type III consists of four different DNA histogram subtypes (Fig. 4). Type II1A has two peaks around the diploid and near-triploid regions. A few cells are found beyond the tetraploid to octaploid region. Type IIIB shows a tetramodal distribution pattern. Two main peaks are located in the diploid and near-6c regions, and two concomitant duplication peaks of the 2c and near-6c stem line are seen in the tetraploid and near-12c regions, respectively. This DNA distribution pattern clearly indicates the existence of two independently proliferating tumor clones within the same tumor mass. Type IIIC tumors contain two cell populations with DNA content at hypotriploidy and tetraploidy. Only a few cells deviate beyond the 4c modal value. Type HID shows a bimodal distribution histogram with two peaks in the near-4c and near-6c regions. Some nuclei are present in the diploid and near-12c regions. Frequency of DNA Histogram Types Table 4 summarizes the relative frequency of DNA histogram types in all patients TYPE II 2C 4C 8C I6C 2C 4C 8C I6C D N A D N A Fig. 3: DNA distribution patterns of the four subtypes in Type II (triploid type). 32C

8 438 OZAKI et al. Jpn. J. Clin. Oncol. December 1981 TYPE Fig. 4: type). DNA Type IIIA I1IB me HID rv-. 2C 4C 8C 2C 4C D N A D N A DNA distribution patterns of the four subtypes in Type III (mixed Table 3 Modal DNA Values in Type III Histograms Modal DNA Values (2c units) & 2.8 & & 6.6 & & & & & & 3.8 & 5.6 & 7.0 Histopathological Diagnosis Large cell ca. Large cell ca. 8C Case No I6C

9 Vol. 11, No. 3 DNA HISTOGRAM TYPES 439 malignancy Type IA- (2c) -Type IB (2c) Type IC (2c & 4c) Type IIIA- (2c s 3c) 1 -Type ID- (4c) Type IIA- (3c) Type IE (4c s 8c) -Type IIB- (3c s 6c). -Type IF (8c) - Type IIC- (5c) 'Type I ID (5c & 10c) ( ): Modal DNA values Fig. 5: tumors. Type IIIB' (2c S 6c) Type IIIC- (3c s 4c) Type HID (4c s 6c) DNA distribution patterns and the grade of malignancy in lung Table 4 The Occurrence of DNA Histogram Types in 70 Patients A B C D E F Total (%) Case No. 50 I (38) Type II _ 34 (49) Til (13) examined. Triploid type (Type II) predominated, with 49%, followed by diploidtetraploid type (Type 1), with 38%. Mixed type (Type III) histograms were noted in only 13% of the patients. This table also reveals that, among the 14 DNA histogram subtypes, subtype A of the triploid type (Type IIA) was most frequently observed. Comparison of DNA Histogram Types in Tumors and their Metastases Comparative analysis of DNA distribution patterns was performed in five patients in Table 5 Comparison of DNA Histogram Subtypes in Tumors and their Metastases Location Histogram Subtype IIA IIA Modal Values (2c units) Histopathological Diagnosis 119 IIIB IIIB IIIB & & & IIA IIA 142 ID ID HA IIB & 6.4

10 440 OZAKI et al. Jpn. J. Clin. Oncol. December 1981 whom primary and metastatic lung tumors were found simultaneously at the time of surgical operation (Table 5). In all cases, histopathological diagnosis of a metastasis was the same as that of its primary tumor. There was one patient in whom the DNA distribution pattern changed from Type IIA in the primary tumor to Type IIB in its metastasis. However, in the other cases, the DNA distribution patterns of the primary tumor and its metastasis were not different. Discussion The validity of measuring nuclear DNA content for ploidy analysis of tumor cells was established by earlier studies with single-cell microspectrophotometry (Atkin, 1954; Leuchtenberger et al., 1954; Sandritter et al., 1966). A modal DNA value determined from the distribution of nuclear DNA content, therefore, represents DNA ploidy of a tumor stem line and is most suitable and reliable as a cytofluorometric parameter for the classification of DNA distribution histograms. Usually a modal DNA value is graphically obtained from a histogram of the frequency of cells containing various DNA contents at arbitrarily chosen intervals. In the present study, modal DNA values were analyzed from the distribution histograms of the logarithmically transformed nuclear DNA content (See Fig. 1). Logarithmical transformation of DNA values permits clearer visualization of a modal pattern, particularly in the higher DNA content region, than does an arbitrarily chosen interval method (Okagaki and Izuo, 1978). The present cytophotometric investigation utilizing the analysis of modal DNA values (DNA ploidy of tumor stem lines) revealed that the nuclear DNA distribution histograms in human lung tumors could be divided into three major types, namely, diploidtetraploid type (Type I), triploid type (Type II), and mixed type (Type III). Each of the three major types was further divided into six, four, and four different DNA histogram subtypes, respectively, according to the modal DNA values. The present finding that subtype A of the triploid type (Type IIA) was most frequently observed in lung tumors coincides with previous reports in the literature (Bohm and Sandritter, 1975; Barlogie et al., 1980). Analysis of the relationship between histopathological grading and DNA modal pattern was not an aim in the present study, largely because lung carcinomas other than adenocarcinomas were too few to compare meaningfully. Changes in DNA ploidy of tumor cell populations along with the progression of neoplasia were discussed in the review by Bohm and Sandritter (1975). A recent flow cytometric study (Barlogie et al., 1978) revealed a change in DNA ploidy from diploidy to triploidy in a patient suffering from immunoblastic lymphadenopathy which subsequently developed into immunoblastic sarcoma. Among the 14 DNA histogram subtypes analyzed in the present study, Types IC, IE, IIB, and IID are all polyploid distribution patterns with two concurrent modes in which the relative DNA values in individual peaks differ from each other by a factor of two (See Tables 1 and 2). This seems to suggest that a tumor cell population in either the 4c, near-6c, or 10c region is created through endoamitosis (Barlogie et al., 1980) or polyploidization (Nakanishi and Fujita, 1977) of the tumor stem line with a basic DNA value at either 2c, near- 3c, or 5c, respectively. In one of five patients whose primary and metastatic lung tumors were examined simultaneously, the DNA distribution pattern was found to change from Type IIA in the primary tumor to Type IIB in its metastasis (See Table 5). Zank and Krug (1970) observed a similar change in DNA distribution pattern between primary and metastatic lung tumors in one case. The nuclear DNA distribution patterns in malignant tumors were found to reflect well the grade of malignancy and the prognosis

11 Vol. II, No. 3 DNA HISTOGRAM TYPES 441 of cancer (Bohm and Sandritter, 1975; Caspersson, 1979). Bichel et al. (1977) reported that, in human prostatic carcinomas, the frequency of tetraploid and octaploid cell populations increased along with the advancement of anaplasia. In a retrospective study on breast carcinomas by Auer et al. (1980), a distinct correlation between different types of DNA histograms and the grade of malignancy was demonstrated. In the present study undertaken with a prospective approach, however, not all of the clinical features of all patients examined are obtainable at this moment, and this incompleteness of clinical information makes us unable to explore thoroughly the diagnostic and prognostic implications of DNA histogram types or subtypes analyzed in this study. However, even when only the clinical information available at present is utilized, the following tendency is recognizable: Type ID, IE, IF, IIA, I1B, IIC, IID, IIIB, II1C, and HID lung tumors are clinically more malignant than Type IA, IB, IC, and IIIA tumors. Our preliminary data in a retrospective research of cancer cells dissociated enzymatically from paraffin embedded specimens also reveal that the prognosis of Type ID, 1IB, or IIIC lung tumors is without exception worse than that of Type IB or IC tumors (unpublished observation). Our current idea for interpreting the diagnostic and prognostic implications of DNA distribution patterns may be depicted as shown in Fig. 5. In order to verify this idea, a new series of retrospective studies for the analysis of the relationship between the DNA modal pattern and the malignancy grade of lung tumor, using the patient's survival time as an indicator, is underway. Acknowledgments We gratefully acknowledge the technical assistance of Kumiko Kawakami and Asae Kawakami. References Adams, L. R. and S. E. Dahlgren, Acta Pathul Microbiol Scand 72: 561, Atkin, N. B., Cancer 17: 1391, Atkin, N. B., Acta Cytol 8: 68, Atkin, N. B., Br Med J 86: 271, Atkin, N. B. and R. Kay, Br J Cancer 40: 210, Auer, G. U., T. O. Caspersson and A. S. Wallgren, Anal Quant Cytol 2: 161, Barlogie, B., B. Drewinko, J. Schumann, W. Gohde, G. Dosik, J. Latreille, D. A. Johnston and E. J. Freireich, Am J Med 69: 195, Barlogie, B., W. Gohde, D. A. Johnston, L. Smallwood, J. Schumann, B. Drewinko and E. J. Freireich, Cancer Res 38: 3333, Bichel, P., P. Frederiksen, T. Kjaer, P. Thommesen and L. L. Vindel0v, Cancer 40: 1206, Bohm, N. and W. Sandritter, Curr Top Pathol 60: 151, Bohm, N., E. Sprenger, G. Schluter and W. Sandritter, Histochemie 15: 194, Caspersson, T. O., Cancer Res 39: 2341, Greisen, O., Acta Otolaryng [Suppl] (Stockh) 276: 1, Kreicbergs, A., A. Zetterberg and G. Soderberg, Anal Quant Cytol 2: 272, Leuchtenberger, C, R. Leuchtenberger and A. M. Davis, Am J Pathol 30: 65, Nakanishi, K. and S. Fujita, Cell Struc Func 2: 261, Nasiell, M., H. Kato, G. Auer, A. Zetterberg, V. Roger and L. Karlen, Cancer 41: 1511, Okagaki, T. and M. Izuo, J Natl Cancer Inst 60: 1251, Sandritter, W., M. Carl and W. Ritter, Acta Cytol 10: 26, Takamatsu, T., K. Nakanishi, Z. Onouchi, M. Fukuda and S. Fujita, Histochemistry 66: 169, Wied, G. L., A. M. Messina and E. Rosenthal, Acta Cytol 10: 31, Zank, M. and H. Krug, Arch Geschwulstforsch 36: 343, 1970.